Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated protein that regulates diverse cellular functions including proliferation, adhesion, migration, differentiation, matrix production and survival. In a study examining differential gene expression in bone from normal and osteopetrotic rats, we discovered that CTGF mRNA was expressed in normal bone. Subsequent studies demonstrated that CTGF is produced and secreted by osteoblasts. Recombinant rat CTGF (rCTGF) was generated and used to examine its effects on osteoblasts in culture. In addition to its effect as a mitogen, rCTGF stimulated functions associated with osteoblast differentiation including alkaline phosphatase activity, osteocalcin gene expression and mineralization. Using a local injection model to test its anabolic effect in vivo, rCTGF induced a potent osteogenic response. The finding that CTGF knockout mice have a skeletal phenotype including severe osteopenia highlights the important role that this growth factor plays in osteoblast development and bone formation. We hypothesize that CTGF is an anabolic bone growth factor. We propose that CTGF is synthesized and secreted by osteoblasts, where it acts as an ECM-associated signaling molecule or a soluble factor to promote osteoblast differentiation and function in an autocrine fashion. Experiments proposed in this application will focus on the association between CTGF and bone with the following aims. Experiments in aim 1 will provide a more comprehensive evaluation of the effects of rCTGF on osteoblast development and function in vitro and bone formation in vivo. Studies in aim 2 will characterize rCTGF binding to osteoblasts and determine whether integrins serve as receptors for CTGF on osteoblasts. The morphological, cytoskeletal and signaling responses of osteoblasts adhered to immobilized rCTGF will also be examined. In aim 3, studies will examine whether specific effects of TGF-beta1 and BMP-2 on osteoblast proliferation/differentiation are CTGF-dependent, and determine whether selected regulatory elements of the CTGF promoter are necessary for transcriptional activation by TGF-beta1 and/or BMP- 2. Experiments in the first part of aim 4 will characterize the skeletal phenotype in CTGF null (-/-) mice and examine the ability of -/- osteoblasts to differentiate in vitro. In the final part of this aim we propose to generate transgenic mice expressing CTGF under control of the osteocalcin promoter and evaluate whether osteoblast-specific expression has an anabolic effect on bone. The identification of a novel anabolic agent in bone and its mechanisms of action will eventually lead to the development of new therapeutic strategies to selectively stimulate osteogenesis in diseases associated with bone loss and in fracture repair.